The goal of this proposal is to investigate the timing and control of cellular senescence in single human cells. Senescence is a state of permanent cell cycle arrest and an inherent defense against tumor progression. Exploiting senescence for therapeutic gain will require a better understanding of basic senescence biology, novel experimental tools to study the phenotype at single-cell resolution, and computational approaches to understand how cells integrate multiple senescence signals. This proposal provides a career transition plan for Dr. Jeremy Purvis that will equip him with the additional training necessary to study senescence at single-cell resolution and form the foundation for developing treatments that induce premature senescence in cells with defective stress responses. During the mentored phase of the award (K99), he will develop a live-cell imaging system for monitoring DNA damage-induced senescence in real time and use this system to determine how upstream signaling of the tumor suppressor p53 control expression of senescence markers (Aim 1). This critical phase of training will be co-supervised by Dr. Galit Lahav and Dr. Peter Sorger (Harvard Medical School), who are both experts in time-lapse microscopy and cell fate decision processes. During the transition to independence (K99/R00), he will identify transcriptional regulators that initiate expression of the key senescence regulato p16INK4a and characterize the dynamics of this transition (Aim 2). The final step in achieving independence (R00) will involve transferring these tools and concepts to study senescence in primary cells lines, focusing specifically on how multiple signals are integrated to achieve a senescence decision (Aim 3). This three-phase transition plan will illuminate our basic understanding of cellular senescence and provide an extensible computational/experimental platform for identifying therapies that induce premature senescence in cancer cells. These aims are highly congruent with the NIGMS's primary goal of supporting basic discoveries that advance the treatment and prevention of human disease. PUBLIC HEALTH RELEVANCE: Cellular senescence is a natural biological process that prevents cells from dividing; in some cases, senescence stops the progression of cancer. The goal of this project is to understand how individual cells enter senescence by observing this transition in real time.